Mass spectrometry assay for congenital adrenal hyperplasia

ABSTRACT

Methods are provided for detecting the amount of one or more CAH panel analytes (i.e., pregnenolone, 17-OH pregnenolone, progesterone, 17-OH progesterone, dehydroepiandrosterone (DHEA), androstenedione, testosterone, deoxycorticosterone, 11-deoxycortisol, and cortisol) in a sample by mass spectrometry. The methods generally involve ionizing one or more CAH panel analytes in a sample and quantifying the generated ions to determine the amount of one or more CAH panel analytes in the sample. In methods where amounts of multiple CAH panel analytes are detected, the amounts of multiple analytes are detected in the same sample injection.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/588,806, filed Sep. 30, 2019, now U.S. Pat. No. 10,697,980, which isa continuation of U.S. application Ser. No. 15/925,294, filed Mar. 19,2018, now U.S. Pat. No. 10,429,396, which is a continuation of U.S.application. Ser. No. 15/401,797, filed Jan. 9, 2017, now U.S. Pat. No.9,921,231, which is a continuation of U.S. application. Ser. No.14/739,864, filed Jun. 15, 2015, now U.S. Pat. No. 9,541,562, which is acontinuation of U.S. application. Ser. No. 14/190,270, filed Feb. 26,2014, now U.S. Pat. No. 9,057,732, which is a continuation of U.S.application. Ser. No. 14/026,793, filed Sep. 13, 2013, now U.S. Pat. No.8,674,291, which is a continuation of U.S. application. Ser. No.13/851,621, filed Mar. 27, 2013, now abandoned, which is a continuationof U.S. application. Ser. No. 13/417,093, filed Mar. 9, 2012, now U.S.Pat. No. 8,415,616, which is a continuation of U.S. application Ser. No.12/645,393, filed Dec. 22, 2009, now U.S. Pat. No. 8,153,962, whichclaims priority to U.S. Provisional Application. No. 61/140,824, filedDec. 24, 2008, the contents of each of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods for measurement of certain analytesthat may indicate congenital adrenal hyperplasia, in particular bytandem mass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Congenital Adrenal Hyperplasia (CAH) is a family of inherited disordersaffecting the adrenal glands. The most common form is 21-hydroxylasedeficiency (21-OHD), which is inherited in severe or mild forms. Thesevere form, called Classical CAH, is usually detected in the newbornperiod or in early childhood. The milder form, called Non-classical CAH(NCAH), may cause symptoms at anytime from infancy through adulthood.NCAH is a much more common disorder than Classical CAH. Fortunately, CAHcan be managed with medication and, with adequate care, affectedindividuals go on to live normal lives.

Cortisol is a steroid produced by the adrenal glands. Cortisol is usedin the body to respond to physical and emotional stress, and maintainadequate energy supply and blood sugar levels.

The adrenal glands are controlled by the pituitary gland, a smallpea-sized gland at the base of the brain. In health individuals, thepituitary gland releases adrenocorticotropic hormone (ACTH) when thereis insufficient cortisol present in the bloodstream. ACTH stimulates theadrenals to produce more cortisol. However, those with CAH haveinsufficient amounts of the enzyme 21-hydroxylase, which is needed toconvert the precursor 17-hydroxyprogesterone (17-OHP) into cortisol. Asa result, the pituitary gland continues to sense the need for cortisoland pumps out more ACTH. This leads to an overabundance of 17-OHP, whichis then converted in the adrenals into excess androgens (masculinizingsteroid hormones).

As such, an individual may be diagnosed with CAH by determining thecirculating levels of the affected steroid hormones. Additionally, anindividual with CAH may be monitored by tracking circulating levels ofthese hormones.

Detection of various affected hormones, either alone (see, e.g., U.S.Pat. No. 7,348,137 (Caulfield, et al.), and U.S. Pat. No. 6,977,143(Caulfield, et al.) describing detection of testosterone by massspectrometric techniques; and U.S. patent application Ser. No.12/207,482 (Ghoshal, et al.) describing detection ofdehydroepiandrosterone (DHEA) by mass spectrometric techniques), or aspart of a multi-analyte panel, have been disclosed in the art. Forexample, Carvalho, V., et al., Chromatogr A 2008, 872:154-61, reported ahormone panel which includes cortisol, 17-OH-progesterone,deoxycorticosterone, and 11-deoxycortisol by tandem mass spectrometry inserum; Guo, T., et al., Clinica Chimica Acta 2006, 372:76-82 reported apanel which includes cortisol, 11-deoxycortisol, androstenedione,testosterone, 17-OH-progesterone, DHEA, and progesterone in serum byHPLC-tandem mass spectrometry. Rauh, M., et al., Steroids 2006,71:450-8, reported a panel which includes 17-OH progesterone,androstenedione, and testosterone by LC-tandem mass spectrometry for thediagnosis and monitoring of hyperandrogenic disorders. Janzen, M., etal., J. Clin Endocrinol Metab 2007, 92:2581-9, reported detection of apanel which included androstenedione, cortisol, 11-deoxycortisol, and17-OH progesterone by tandem mass spectrometric techniques. Kushnir, M.,et al., Clinical Chemistry 2006, 52:1559-67, reported detection of apanel which included derivatized forms of 11-deoxycortisol, 17-OHprogesterone, 17-OH pregnenolone, and pregnenolone in blood by LC-tandemmass spectrometric techniques. Lacey, et al., Clinical Chemistry 2004,50:621-5, and Minutti, C., et al., J. Clin Endocrinol Metab 2004,89:3687-93, both reported detection of panels which included 17-OHprogesterone, androstenedione, and cortisol by tandem mass spectrometrictechniques. Shindo, N., et al., Biomedical Chromatogr 1990, 4:171-4,reported detection of a panel which included 17-OH progesterone,11-deoxycortisol, progesterone, cortisol, testosterone, 17-OHpregnenolone and pregnenolone by plasmaspray LC-MS.

SUMMARY OF THE INVENTION

Methods are provided for detecting the amount of one or more CAH panelanalytes (i.e., pregnenolone, 17-OH pregnenolone, progesterone, 17-OHprogesterone, dehydroepiandrosterone (DHEA), androstenedione,testosterone, deoxycorticosterone, 11-deoxycortisol (also known as11-desoxycortisol), cortisone, corticosterone, dihydrotestosterone, andcortisol) in a sample by mass spectrometry, including tandem massspectrometry. In methods where amounts of multiple CAH panel analytesare detected, the amounts of multiple analytes are detected in the samesample injection.

These methods include: subjecting a sample, purified by methodsdescribed below, to an ionization source under conditions suitable toproduce one or more ions detectable by mass spectrometry; determiningthe amounts of one or more ions from each of the one or more analytes bytandem mass spectrometry; and using the amounts of one or more ions fromeach of the one or more analytes to determine the amount of each one ormore analyte in the sample. Preferably, the sample is a biologicalfluid; more preferably the sample is serum.

In some embodiments, the methods include detecting one or more analytesselected from the group consisting of pregnenolone and 17-OHpregnenolone. In some embodiments where the one or more analytescomprises pregnenolone, the one or more ions from pregnenolone compriseone or more ions selected from the group consisting of ions with a massto charge ratio of 299.2, 105.6, and 91.1. In some embodiments where theone or more analytes comprises 17-OH pregnenolone, the one or more ionsfrom 17-OH pregnenolone comprise one or more ions selected from thegroup consisting ions with a mass to charge ratio of 297.2±0.5,105.6±0.5, and 91.1±0.5. In some embodiments, both pregnenolone and17-OH pregnenolone are determined.

In some embodiments, the methods also include (in addition topregnenolone and 17-OH pregnenolone) determining the amount of one ormore additional analytes in the sample selected from the groupconsisting of cortisol, cortisone, corticosterone, 11-deoxycortisol,testosterone, dehydroepiandrosterone (DHEA), deoxycorticosterone,androstenedione, 17-OH progesterone, and progesterone.

In preferred embodiments of the present invention, the amounts of two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, ten or more, eleven or more, twelveor more, or thirteen CAH panel analytes are determined.

In some embodiments, the levels of two or more CAH panel analytes aredetermined and at least one of the two or more analytes is selected fromthe group consisting of pregnenolone and 17-OH pregnenolone.

In some embodiments, one of two or more analytes is deoxycorticosterone,and a second of two or more analytes is selected from the groupconsisting of pregnenolone, 17-OH pregnenolone, progesterone,dehydroepiandrosterone (DHEA), androstenedione, and testosterone. Insome embodiments where deoxycorticosterone is determined, the one ormore ions from deoxycorticosterone comprise one or more ions selectedfrom the group of ions with a mass to charge ratio of 331.2±0.5,109.5±0.5, and 97.1±0.5.

In some embodiments, the sample has been purified by liquidchromatography prior to being subjected to an ionization source. In someembodiments, liquid chromatography may include one or more of highperformance liquid chromatography, ultra high performance liquidchromatography, and turbulent flow liquid chromatography. In someembodiments, liquid chromatography is a combination of turbulent flowliquid chromatography and either high performance liquid chromatographyor ultra high performance liquid chromatography.

In some embodiments, the one or more detected analytes are selected fromthe group consisting of pregnenolone, 17-OH pregnenolone, progesterone,17-OH progesterone, dehydroepiandrosterone (DHEA), androstenedione,testosterone, deoxycorticosterone, 11-deoxycortisol, and cortisol,wherein if one or more detected analytes is only one analyte, the oneanalyte is not DHEA or testosterone. In these embodiments, the methodincludes the additional step of subjecting said sample to turbulent flowliquid chromatography to obtain a sample enriched in said one or moreanalytes subject to determination.

In a second aspect, methods are presented for diagnosing congenitaladrenal hyperplasia (CAH), or some other condition affecting productionof adrenal hormones. The methods for diagnosing include obtaining asample of a body fluid from an individual suspected of having CAH anddetermining the level of two or more CAH panel analytes by tandem massspectrometry.

In some of the CAH diagnosing embodiments, one of the two or more CAHpanel analytes are selected from the group consisting of 17-OHpregnenolone, 17-OH progesterone, dehydroepiandrosterone (DHEA),androstenedione, deoxycorticosterone, and 11-deoxycortisol. In somerelated embodiments, the methods further include determining the ratioof the levels of one CAH panel analyte to another CAH panel analyte. Insome embodiments, the ratio of the levels of 17-OH pregnenolone to 17-OHprogesterone is determined. In the ratio of the levels of DHEA toandrostenedione is determined.

Some embodiments of these methods may be used to diagnose a21-hydroxylase deficiency form of CAH by determining an increased levelof 17-OH progesterone over the level in a comparable body fluid samplefrom an individual without a 21-hydroxylase deficiency form of CAH. Someembodiments of these methods may be used to diagnose a11-beta-hydroxylase deficiency form of CAH by determining increasedlevels of 11-deoxycortisol and deoxycorticosterone over the levels in acomparable body fluid sample from an individual without a11-beta-hydroxylase deficiency form of CAH.

In some embodiments, one of the CAH panel analytes is pregnenolone. Inrelated embodiments, one or more ions from pregnenolone comprise ionsselected from the group consisting of ions with a mass to charge ratio(m/z) of 299.2±0.5, 105.6±0.5, and 91.1±0.5. In particularly preferredembodiments, one or more ions from pregnenolone comprise a precursor ionwith m/z of 299.2±0.5, and one or more fragment ions selected from thegroup of ions with m/z of 105.6±0.5, and 91.1±0.5.

In some embodiments, one of the CAH panel analytes is 17-OHpregnenolone. In related embodiments, one or more ions from 17-OHpregnenolone comprise ions selected from the group consisting of ionswith a mass to charge ratio (m/z) of 297.2±0.5, 105.6±0.5, and 91.1±0.5.In particularly preferred embodiments, one or more ions from 17-OHpregnenolone comprise a precursor ion with m/z of 297.2±0.5, and one ormore fragment ions selected from the group of ions with m/z of 105.6±0.5and 91.0±0.5.

In some embodiments, one of the CAH panel analytes is progesterone. Inrelated embodiments, one or more ions from progesterone comprise ionsselected from the group consisting of ions with a mass to charge ratio(m/z) of 315.2±0.5, 109.1±0.5, and 97.1±0.5. In particularly preferredembodiments, one or more ions from progesterone comprise a precursor ionwith m/z of 315.2±0.5, and one or more fragment ions selected from thegroup of ions with m/z of 109.1±0.5 and 97.1±0.5.

In some embodiments, one of the CAH panel analytes is 17-OHprogesterone. In related embodiments, one or more ions from 17-OHprogesterone comprise ions selected from the group consisting of ionswith a mass to charge ratio (m/z) of 331.0±0.5, 109.0±0.5, and 96.9±0.5.In particularly preferred embodiments, one or more ions from 17-OHprogesterone comprise a precursor ion with m/z of 331.0±0.5, and one ormore fragment ions selected from the group of ions with m/z of 109.0±0.5and 96.9±0.5.

In some embodiments, one of the CAH panel analytes is DHEA. In relatedembodiments, one or more ions from DHEA comprise ions selected from thegroup consisting of ions with a mass to charge ratio (m/z) of 253.1±0.5,197.1±0.5, and 157.1±0.5. In particularly preferred embodiments, one ormore ions from DHEA comprise a precursor ion with m/z of 253.1±0.5, andone or more fragment ions selected from the group of ions with m/z of197.1±0.5 and 157.1±0.5.

In some embodiments, one of the CAH panel analytes is androstenedione.In related embodiments, one or more ions from androstenedione compriseions selected from the group consisting of ions with a mass to chargeratio (m/z) of 287.1±0.5, 109.1±0.5, and 91.1±0.5. In particularlypreferred embodiments, one or more ions from androstenedione comprise aprecursor ion with m/z of 287.1±0.5, and one or more fragment ionsselected from the group of ions with m/z of 109.1±0.5, and 91.1±0.5.

In some embodiments, one of the CAH panel analytes is testosterone. Inrelated embodiments, one or more ions from testosterone comprise ionsselected from the group consisting of ions with a mass to charge ratio(m/z) of 289.1±0.5, 109.0±0.5, and 97.0±0.5. In particularly preferredembodiments, one or more ions from testosterone comprise a precursor ionwith m/z of 289.1±0.5, and one or more fragment ions selected from thegroup of ions with m/z of 109.0±0.5 and 97.0±0.5.

In some embodiments, one of the CAH panel analytes isdeoxycorticosterone. In related embodiments, one or more ions fromdeoxycorticosterone comprise ions selected from the group consisting ofions with a mass to charge ratio (m/z) of 331.2±0.5, 109.5±0.5, and97.1±0.5. In particularly preferred embodiments, one or more ions fromdeoxycorticosterone comprise a precursor ion with m/z of 331.2±0.5, andone or more fragment ions selected from the group of ions with m/z of109.5±0.5 and 97.1±0.5.

In some embodiments, one of the CAH panel analytes is 11-deoxycortisol(also known as 11-desoxycortisol). In related embodiments, one or moreions from 11-deoxycortisol comprise ions selected from the groupconsisting of ions with a mass to charge ratio (m/z) of 347.1±0.5,109.1±0.5, and 97.1±0.5. In particularly preferred embodiments, one ormore ions from 11-deoxycortisol comprise a precursor ion with m/z of347.1±0.5, and one or more fragment ions selected from the group of ionswith m/z of 109.1±0.5 and 97.1±0.5.

In some embodiments, one of the CAH panel analytes isdihydrotestosterone. In related embodiments, one or more ions fromdihydrotestosterone comprise ions selected from the group consisting ofions with a mass to charge ratio (m/z) of 273.2±0.5, 105.1±0.5, and91.1±0.5. In particularly preferred embodiments, one or more ions fromdihydrotestosterone comprise a precursor ion with m/z of 273.2±0.5, andone or more fragment ions selected from the group of ions with m/z of105.1±0.5 and 91.1±0.5.

In some embodiments, one of the CAH panel analytes is corticosterone. Inrelated embodiments, one or more ions from corticosterone comprise ionsselected from the group consisting of ions with a mass to charge ratio(m/z) of 347.2±0.5, 121.1±0.5, and 91.1±0.5. In particularly preferredembodiments, one or more ions from corticosterone comprise a precursorion with m/z of 347.2±0.5, and one or more fragment ions selected fromthe group of ions with m/z of 121.1±0.5 and 91.1±0.5.

In some embodiments, one of the CAH panel analytes is cortisone. Inrelated embodiments, one or more ions from cortisone comprise ionsselected from the group consisting of ions with a mass to charge ratio(m/z) of 361.1±0.5, 163.2±0.5, and 105.1±0.5. In particularly preferredembodiments, one or more ions from cortisone comprise a precursor ionwith m/z of 361.1±0.5, and one or more fragment ions selected from thegroup of ions with m/z of 163.2±0.5 and 105.1±0.5.

In some embodiments, one of the CAH panel analytes is cortisol. Inrelated embodiments, one or more ions from cortisol comprise ionsselected from the group consisting of ions with a mass to charge ratio(m/z) of 363.1±0.5, 121.0±0.5, and 90.9±0.5. In particularly preferredembodiments, one or more ions from cortisol comprise a precursor ionwith m/z of 363.1±0.5, and one or more fragment ions selected from thegroup of ions with m/z of 121.0±0.5 and 90.9±0.5.

Embodiments of the present invention may involve the combination ofliquid chromatography with mass spectrometry. In some embodiments, theliquid chromatography may comprise HPLC, UPLC, TFLC, or any combinationthereof. For example, in some embodiments, HPLC, alone or in combinationwith one or more purification methods such as for example SPE (e.g.,TFLC) and/or protein precipitation and filtration, is utilized to purifyan analyte in a sample. In other embodiments, the liquid chromatographymay comprise UPLC, either alone or in combination with one or moreadditional purification methods, such as SPE (e.g., TFLC) and/or proteinprecipitation and filtration, to purify an analyte in a sample.

In some embodiments, at least one purification step and massspectrometric analysis is conducted in an on-line fashion. In anotherpreferred embodiment, the mass spectrometry is tandem mass spectrometry(MS/MS).

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention.

In preferred embodiments, one or more separately detectable internalstandards is provided in the sample, the amount of which is alsodetermined in the sample. An internal standard may be used to accountfor loss of analytes during sample processing in order to get a moreaccurate value of a measured analyte in the sample. In theseembodiments, all or a portion of one or more endogenous analytesselected from the group consisting of CAH panel analytes, and the one ormore internal standards present in the sample are ionized to produce aplurality of ions detectable in a mass spectrometer. In preferredembodiments, the amount of ions generated from an analyte of interestmay be related to the presence of amount of analyte of interest in thesample by comparison to one or more internal standards.

Preferred internal standards include d₅-testosterone, d₄-cortisol, andd₉-progestrone. However, these preferred internal standards is notintended to be exclusive, i.e., other suitable internal standards may beused. Isotopically labeled analogues of CAH panel analytes, such asd₂-11-deoxycortisol, d₇-androstenedione, d₈-17-OH progesterone,d₂-testosterone, and d₂-DHEA are also useful for use as internalstandards.

In other embodiments, the amount of an analyte in a sample may bedetermined by comparison of the amount of one or more analyte ionsdetected by mass spectrometry to the amount of one or more standard ionsdetected by mass spectrometry in an external reference standard.Exemplary external reference standards may comprise blank plasma orserum spiked with a known amount of one or more of the above describedinternal standards and/or analytes of interest.

The features of the embodiments listed above may be combined withoutlimitation for use in methods of the present invention.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium,¹³C, and ¹⁵N. Deuterium is a useful label because it can potentiallyproduce three mass unit shifts in a labeled methylation product relativeto an unlabeled methylation product. For example, d₅-testosterone has amass five mass units higher than testosterone. An isotopic label may beincorporated at one or more positions in the molecule and one or morekinds of isotopic labels may be used on the same isotopically labeledmolecule.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “sample” refers to any sample that may containan analyte of interest. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like. Preferred samples for use in thepresent invention comprise human serum.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (also sometimes known as “high pressure liquid chromatography”)refers to liquid chromatography in which the degree of separation isincreased by forcing the mobile phase under pressure through astationary phase, typically a densely packed column. As used herein, theterm “ultra high performance liquid chromatography” or “UPLC” or “UHPLC”(sometimes known as “ultra high pressure liquid chromatography”) refersto HPLC which occurs at much higher pressures than traditional HPLCtechniques.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow.” When fluid flowsslowly and smoothly, the flow is called “laminar flow.” For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns,” which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%.

As used herein, the term “on-line” or “inline,” for example as used in“on-line automated fashion” or “on-line extraction,” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “sample injection” refers to introducing analiquot of a single sample into an analytical instrument, for example amass spectrometer. This introduction may occur directly or indirectly.An indirect sample injection may be accomplished, for example, byinjecting an aliquot of a sample into a HPLC or UPLC analytical columnthat is connected to a mass spectrometer in an on-line fashion.

As used herein, the term “same sample injection” with respect tomultiple analyte analysis by mass spectrometry means that the ions fortwo or more different analytes are determined essentially simultaneouslyby measuring ions for the different analytes from the same (i.e.identical) sample injection.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, and triethanolamineThe choice of an appropriate matrix for a compound or sample is anempirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photoionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photoionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatscauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-M shows plots of elution profiles generated by HPLC-MS/MSanalysis of various CAH panel analytes (cortisol, cortisone,corticosterone, 11-deoxycortisol, testosterone, DHEA,deoxycorticosterone, androstenedione, 17-OH progesterone, progesterone,dihydrotestosterone, pregnenolone, and 17-OH pregnenolone) in a patientsample. Details are discussed in Example 1.

FIG. 2 shows product ion peaks generated from tandem mass spectrometricfragmentation of cortisol (precursor ion of about 363.1±0.5). Detailsare described in Example 2.

FIG. 3 shows product ion peaks generated from tandem mass spectrometricfragmentation of cortisone (precursor ion of about 361.1±0.5). Detailsare described in Example 2.

FIG. 4 shows product ion peaks generated from tandem mass spectrometricfragmentation of corticosterone (precursor ion of about 347.2±0.5).Details are described in Example 2.

FIG. 5 shows product ion peaks generated from tandem mass spectrometricfragmentation of 11-deoxycortisol (precursor ion of about 347.1±0.5).Details are described in Example 2.

FIG. 6 shows product ion peaks generated from tandem mass spectrometricfragmentation of testosterone (precursor ion of about 289.1±0.5).Details are described in Example 2.

FIG. 7 shows product ion peaks generated from tandem mass spectrometricfragmentation of DHEA (precursor ion of about 253.1±0.5). Details aredescribed in Example 2.

FIG. 8 shows product ion peaks generated from tandem mass spectrometricfragmentation of deoxycorticosterone (precursor ion of about 331.2±0.5).Details are described in Example 2.

FIG. 9 shows product ion peaks generated from tandem mass spectrometricfragmentation of androstenedione (precursor ion of about 287.1±0.5).Details are described in Example 2.

FIG. 10 shows product ion peaks generated from tandem mass spectrometricfragmentation of 17-OH progesterone (precursor ion of about 331.0±0.5).Details are described in Example 2.

FIG. 11 shows product ion peaks generated from tandem mass spectrometricfragmentation of progesterone (precursor ion of about 315.2±0.5).Details are described in Example 2.

FIG. 12 shows product ion peaks generated from tandem mass spectrometricfragmentation of dihydrotestosterone (precursor ion of about 273.2±0.5).Details are described in Example 2.

FIG. 13 shows product ion peaks generated from tandem mass spectrometricfragmentation of pregnenolone (precursor ion of about 299.2±0.5).Details are described in Example 2.

FIG. 14 shows product ion peaks generated from tandem mass spectrometricfragmentation of 17-OH pregnenolone (precursor ion of about 297.2±0.5).Details are described in Example 2.

FIGS. 15A-J show results of linearity studies for analysis of CAH panelanalytes by HPLC-MS/MS. Details are described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of one or more CAH panelanalytes in a sample. More specifically, mass spectrometric methods aredescribed for quantifying one or more CAH panel analytes in a samplethat typically has been purified by one or more steps prior to massspectrometry. The methods may utilize a liquid chromatography step suchas HPLC to perform a purification of selected analytes combined withmethods of mass spectrometry (MS) thereby providing a high-throughputassay system for quantifying one or more CAH panel analytes in a sample.The preferred embodiments are particularly well suited for applicationin large clinical laboratories for automated CAH monitoring.

Suitable samples for use in methods of the present invention include anysample that may contain one or more of the analytes of interest. In somepreferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as urine, blood, plasma, serum, saliva, and cerebrospinalfluid, or tissue samples; preferably plasma or serum; most preferablyserum. Such samples may be obtained, for example, from a patient; thatis, a living person, male or female, presenting oneself in a clinicalsetting for diagnosis, prognosis, or treatment of a disease orcondition. The sample is preferably obtained from a patient, forexample, a blood sample, which may be collected from a patient forremoval as plasma or serum.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. Thus, a derivatized analyte is a base molecule that hasbeen reacted with another molecule for the purpose of, for example,facilitating purification, ionization, fragmentation, detection, or anycombination thereof. In the methods described herein, the CAH panelanalytes quantitated by mass spectrometry are preferably notderivatized.

The levels of circulating CAH panel analytes (determined by methods ofthe present invention) may be used to diagnose CAH, or some othercondition affecting production of adrenal hormones, in an individual.The diagnosis of CAH depends is based on inadequate production ofcortisol and aldosterone (or both) in conjunction with elevatedconcentrations of precursor hormones. For example, the 21-hydroxylasedeficiency form of CAH can be detected by a high serum concentration of17-hydroxyprogesterone (usually >1000 ng/dL) and urinary pregnanetriol(metabolite of 17-hydroxyprogesterone) in the presence of clinicalfeatures suggestive of the disease (eg, salt wasting, clitoromegaly orambiguous genitalia [in a female patient], precocious pubic hair,excessive growth, premature phallic enlargement in the absence oftesticular enlargement, hirsutism, oligomenorrhea, female infertility).

In some embodiments, at least one of two or more determined CAH panelanalytes are selected from the group consisting of 17-OH pregnenolone,17-OH progesterone, dehydroepiandrosterone (DHEA), androstenedione,deoxycorticosterone, and 11-deoxycortisol. For example, CAH caused by11-beta-hydroxylase deficiency can be detected by measuring elevatedconcentrations of 11-deoxycortisol and deoxycorticosterone or by anelevation in the ratio of a 24-hour urinary measurement oftetrahydrocompound S (metabolite of 11-deoxycortisol) totetrahydrocompound F (metabolite of cortisol).

In some instances, it may be useful to determine a ratio of the levelsof one CAH panel analyte to another in the sample. For example, 3-β-OHsteroid dehydrogenase deficiency may be indicated by an abnormal ratioof 17-OH pregnenolone to 17-OH progesterone and/or an abnormal ratio ofDHEA to androstenedione. In some embodiments, diagnostic methods of thepresent invention further comprise determining the ratio of the levelsof one CAH panel analyte to another CAH panel analyte in the sample;preferably 17-OH pregnenolone to 17-OH progesterone, or DHEA toandrostenedione. The determined ratios may then be compared to ratios ofthe same analytes in samples from individuals without CAH. Preferablythe comparative samples are from normal, healthy individuals.

As used herein, “abnormal” indicates a state or condition that deviatesfrom that observed a normal, healthy individual. Thus, an abnormal levelor ratio represents a relative condition compared to that level or ratioobserved in a health individual. One of skill in the art would be ableto determine the degree of abnormality required to diagnose CAH in anindividual.

The present invention also contemplates kits for a CAH diagnosis ormonitoring assay. A kit for a CAH diagnosis or monitoring assay mayinclude a kit comprising the compositions provided herein. For example,a kit may include packaging material and measured amounts of one or moreisotopically labeled internal standards, in amounts sufficient for atleast one assay. Typically, the kits will also include instructionsrecorded in a tangible form (e.g., contained on paper or an electronicmedium) for using the packaged reagents for use in a CAH diagnosis ormonitoring assay.

Sample Preparation for Mass Spectrometry

Some or all CAH panel analytes in a sample may be bound to proteins, ifalso present in the sample. Various methods may be used to disrupt theinteraction between CAH panel analytes and protein prior to theimplementation of one or more enrichment steps and/or MS analysis sothat the amount of a CAH panel analyte measured by mass spectrometry isa reflection of the total for that CAH panel analyte in the sample. OnceCAH panel analytes and proteins have been separated in the sample, CAHpanel analytes may be enriched relative to one or more other componentsin the sample (e.g. protein) by various methods known in the art, suchas for example, liquid chromatography, filtration, centrifugation, thinlayer chromatography (TLC), electrophoresis including capillaryelectrophoresis, affinity separations including immunoaffinityseparations, extraction methods including ethyl acetate or methanolextraction, and the use of chaotropic agents or any combination of theabove or the like.

Protein precipitation is one method of preparing a sample, especially abiological sample, such as serum or plasma. Such protein purificationmethods are well known in the art, for example, Polson et al., Journalof Chromatography B 785:263-275 (2003), describes protein precipitationtechniques suitable for use in the methods. Protein precipitation may beused to remove most of the protein from the sample leaving CAH panelanalytes in the supernatant. The samples may be centrifuged to separatethe liquid supernatant from the precipitated proteins. The resultantsupernatant may then be applied to liquid chromatography and subsequentmass spectrometry analysis. In certain embodiments, the use of proteinprecipitation obviates the need for turbulent flow liquid chromatography(TFLC) or other on-line extraction prior to HPLC and mass spectrometry.Accordingly in such embodiments, the method involves (1) performing aprotein precipitation of the sample of interest; and (2) loading thesupernatant directly onto the HPLC-mass spectrometer without usingon-line extraction or turbulent flow liquid chromatography (TFLC).

In other embodiments, CAH panel analytes may be released from a proteinwithout having to precipitate the protein. For example, an aqueousformic acid solution may be added to the sample to disrupt interactionbetween a protein and a CAH panel analyte. Alternatively, ammoniumsulfate or an aqueous solution of formic acid in ethanol may be added tothe sample to disrupt ionic interactions between a carrier protein and aCAH panel analyte without precipitating the carrier protein.

In some preferred embodiments, TFLC, alone or in combination with one ormore purification methods, may be used to purify CAH panel analytesprior to mass spectrometry. In such embodiments CAH panel analytes maybe extracted using an TFLC extraction cartridge which captures theanalytes, then eluted and chromatographed on a second TFLC column oronto an HPLC or UPLC analytical column prior to ionization. Because thesteps involved in these chromatography procedures can be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature can result insavings of time and costs, and eliminate the opportunity for operatorerror.

It is believed that turbulent flow, such as that provided by TFLCcolumns and methods, may enhance the rate of mass transfer, improvingseparation characteristics. TFLC columns separate components by means ofhigh chromatographic flow rates through a packed column containing rigidparticles. By employing high flow rates (e.g., 3-5 mL/min), turbulentflow occurs in the column that causes nearly complete interactionbetween the stationary phase and the analyte(s) of interest. Anadvantage of using TFLC columns is that the macromolecular build-upassociated with biological fluid matrices is avoided since the highmolecular weight species are not retained under the turbulent flowconditions. TFLC methods that combine multiple separations in oneprocedure lessen the need for lengthy sample preparation and operate ata significantly greater speed. Such methods also achieve a separationperformance superior to laminar flow (HPLC) chromatography. TFLC oftenallows for direct injection of biological samples (plasma, urine, etc.).Direct injection is difficult to achieve in traditional forms ofchromatography because denatured proteins and other biological debrisquickly block the separation columns TFLC also allows for very lowsample volume of less than 1 mL, preferably less than 0.5 mL, preferablyless than 0.2 mL, preferably about 0.1 mL.

Examples of TFLC applied to sample preparation prior to analysis by massspectrometry have been described elsewhere. See, e.g., Zimmer et al., J.Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367;5,919,368; 5,795,469; and 5,772,874. In certain embodiments of themethod, samples are subjected to protein precipitation as describedabove prior to loading on the TFLC column; in alternative preferredembodiments, the samples may be loaded directly onto the TFLC withoutbeing subjected to protein precipitation. Preferably, TFLC is used inconjunction with HPLC to extract and purify one or more CAH panelanalytes without subjecting the sample to protein precipitation. Inrelated preferred embodiments, purifying the sample prior to MS analysisinvolves (i) applying the sample to a TFLC extraction column, (ii)washing the TFLC extraction column under conditions whereby one or moreHRT panel analytes are retained by the column, (iii) eluting retainedCAH panel analytes from the TFLC extraction column, (iv) applying theretained material to an analytical column, and (v) eluting purified CAHpanel analytes from the analytical column. The TFLC extraction column ispreferably a large particle column. In various embodiments, one of moresteps of the methods may be performed in an on-line, automated fashion.For example, in one embodiment, steps (i)-(v) are performed in anon-line, automated fashion. In another, the steps of ionization anddetection are performed on-line following steps (i)-(v).

One means of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain LC techniques,including HPLC, rely on relatively slow, laminar flow technology.Traditional HPLC analysis relies on column packings in which laminarflow of the sample through the column is the basis for separation of theanalyte of interest from the sample. The skilled artisan will understandthat separation in such columns is a diffusional process and may selectHPLC instruments and columns that are suitable for use with CAH panelanalytes. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesinclude a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties. One suitablebonded surface is a hydrophobic bonded surface such as an alkyl bondedsurface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18bonded alkyl groups. The chromatographic column includes an inlet portfor receiving a sample directly or indirectly from a solid-phaseextraction or TFLC column and an outlet port for discharging an effluentthat includes the fractionated sample.

In one embodiment, the sample is applied to the column at the inletport, eluted with a solvent or solvent mixture, and discharged at theoutlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted on a hydrophobic columnchromatographic system. In certain preferred embodiments, TFLC and HPLCare performed using HPLC Grade organic and aqueous mobile phases. Insome embodiments, the mobile phase may be 100% acetonitrile or methanol.In some embodiments, the aqueous mobile phase may be Ultra Pure water oran aqueous formic acid solution with a concentration between about 0.1%to about 20% formic acid.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, TFLC may be used for purification of one or moreCAH panel analytes prior to mass spectrometry. In such embodiments, oneor more CAH panel analytes may be extracted using a TFLC extractioncolumn, then eluted and chromatographed on a second TFLC column or ontoan analytical HPLC column prior to ionization. For example, CAH panelanalyte extraction with an TFLC extraction column may be accomplishedwith a large particle size (50 μm) packed column. Sample eluted off ofthis column may then be transferred to an HPLC analytical column forfurther purification prior to mass spectrometry. Because the stepsinvolved in these chromatography procedures may be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized. This feature may result insavings of time and costs, and eliminate the opportunity for operatorerror.

Detection and Quantitation by Mass Spectrometry

In various embodiments, one or more CAH panel analytes may be ionized byany method known to the skilled artisan. Mass spectrometry is performedusing a mass spectrometer, which includes an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electronionization, chemical ionization, electrospray ionization (ESI), photonionization, atmospheric pressure chemical ionization (APCI),photoionization, atmospheric pressure photoionization (APPI), fast atombombardment (FAB), liquid secondary ionization (LSI), matrix assistedlaser desorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, surface enhanced laser desorptionionization (SELDI), inductively coupled plasma (ICP) and particle beamionization. The skilled artisan will understand that the choice ofionization method may be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

The one or more CAH panel analytes may be ionized in positive ornegative mode to create one or more CAH panel ions. In some embodiments,the one or more CAH panel analytes are ionized by electrosprayionization (ESI) in positive or negative mode; preferably positive mode.In alternative embodiments, the one or more CAH panel analytes areionized by atmospheric pressure chemical ionization (APCI) in positiveor negative mode; preferably positive mode. In related preferredembodiments, the one or more CAH panel ions are in a gaseous state andthe inert collision gas is argon or nitrogen.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass-to-charge ratio. Suitable analyzers fordetermining mass-to-charge ratios include quadrupole analyzers, iontraps analyzers, and time-of-flight analyzers. Exemplary ion trapmethods are described in Bartolucci, et al., Rapid Commun. MassSpectrom. 2000, 14:967-73.

The ions may be detected using several detection modes. For example,selected ions may be detected, i.e. using a selective ion monitoringmode (SIM), or alternatively, ions may be detected using a scanningmode, e.g., multiple reaction monitoring (MRM) or selected reactionmonitoring (SRM). Preferably, the mass-to-charge ratio is determinedusing a quadrupole analyzer. For example, in a “quadrupole” or“quadrupole ion trap” instrument, ions in an oscillating radio frequencyfield experience a force proportional to the DC potential appliedbetween electrodes, the amplitude of the RF signal, and the mass/chargeratio. The voltage and amplitude may be selected so that only ionshaving a particular mass/charge ratio travel the length of thequadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of one or moreCAH analytes. Methods of generating and using such standard curves arewell known in the art and one of ordinary skill is capable of selectingan appropriate internal standard. For example, in preferred embodimentsone or more isotopically labeled analogues of CAH panel analytes (e.g.,d₅-testosterone and d₉-progestrone) may be used as internal standards.Numerous other methods for relating the amount of an ion to the amountof the original molecule will be well known to those of ordinary skillin the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation (CAD) isoften used to generate fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In some embodiments, one or more CAH panel analytes are quantified in asample using MS/MS as follows. The samples are subjected to liquidchromatography, preferably TFLC followed by HPLC; the flow of liquidsolvent from the chromatographic column enters the heated nebulizerinterface of an MS/MS analyzer; and the solvent/analyte mixture isconverted to vapor in the heated tubing of the interface. The CAHanalytes contained in the nebulized solvent are ionized by the coronadischarge needle of the interface, which applies a large voltage to thenebulized solvent/analyte mixture. The ions, e.g. precursor ions, passthrough the orifice of the instrument and enter the first quadrupole.Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection ofions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3,respectively) based on their mass to charge ratio (m/z). Quadrupole 2(Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios of one of the CAH panel analytes. Precursor ionswith the correct mass/charge ratios are allowed to pass into thecollision chamber (Q2), while unwanted ions with any other mass/chargeratio collide with the sides of the quadrupole and are eliminated.Precursor ions entering Q2 collide with neutral collision gas moleculesand fragment. The fragment ions generated are passed into quadrupole 3(Q3), where the fragment ions of the selected CAH panel analyte areselected while other ions are eliminated. During analysis of a singlesample injection, Q1 and/or Q3 may be adjusted such that mass/chargeratios of one or more precursor ion/fragment ion pairs specific to oneCAH panel analyte are first selected, followed at some later time by theselection of mass/charge ratios of one or more precursor ion/fragmention pairs specific to a second CAH panel analyte, optionally repeated atsome later time for as many CAH panel analytes as is desired. Inparticularly preferred embodiments, at least one precursor ion/fragmention pair is selected for every CAH panel analyte in an analysis of asingle sample injection, although the sequence of pair selection mayoccur in any order.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of a CAH panel analytethat may be used for selection in quadrupole 3 (Q3). Preferred precursorion/fragment ions for CAH panel analytes and exemplary internalstandards are found in Table 1.

TABLE 1 Preferred Precursor Ion/Fragment Ion Mass to Charge Ratios ofCAH Panel Analytes Analyte Parent (m/z) Fragment(s) (m/z) cortisol 363.1± 0.5 121.1 ± 0.5, 91.1 ± 0.5 cortisone 361.1 ± 0.5 163.2 ± 0.5, 105.1 ±0.5 corticosterone 347.2 ± 0.5 121.1 ± 0.5, 91.1 ± 0.5 11-deoxycortisol347.1 ± 0.5 109.1 ± 0.5, 97.1 ± 0.5 testosterone 289.1 ± 0.5 109.0 ±0.5, 97.0 ± 0.5 DHEA 253.1 ± 0.5 197.1 ± 0.5, 157.1 ± 0.5deoxycorticosterone 331.2 ± 0.5 109.5 ± 0.5, 97.1 ± 0.5 androstenedione287.1 ± 0.5 109.1 ± 0.5, 91.1 ± 0.5 17-OH progesterone 331.0 ± 0.5 109.0± 0.5, 96.9 ± 0.5 progesterone 315.2 ± 0.5 109.1 ± 0.5, 97.1 ± 0.5dihydrotestosterone 273.2 ± 0.5 105.1 ± 0.5, 91.1 ± 0.5 pregnenolone299.2 ± 0.5 105.6 ± 0.5, 91.1 ± 0.5 17-OH pregnenolone 297.2 ± 0.5 105.6± 0.5, 91.1 ± 0.5

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of each CAH panel analyte detected. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte using calibration standard curves basedon peaks of one or more ions of an internal molecular standard.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1: Extraction of CAH Panel Analytes from Samples UsingLC

Liquid chromatography was performed on purified samples made from100-200 μL serum.

A binary HPLC gradient of an aqueous phase (i.e., mobile phase A) and anorganic phase (i.e., mobile phase B) was applied to the analyticalcolumn to separate CAH panel analytes from each other and other analytescontained in the sample. The gradient starts at 85% mobile phase A/15%mobile phase B and ramps to 25% mobile phase A/75% mobile phase B over50 seconds. The approximate retention times of the various CAH panelanalytes are shown in Table 2.

TABLE 2 Approximate Retention Times of CAH Panel Analytes AnalyteApproximate Retention Time (min) cortisol 2.74 cortisone 2.76corticosterone 2.93 11-deoxycortisol 2.97 testosterone 3.37 DHEA 3.54androstenedione 3.54 deoxycorticosterone 3.55 17-OH progesterone 3.55progesterone 4.80 dihydrotestosterone 4.83 pregnenolone 4.83 17-OHpregnenolone 4.84

Exemplary chromatograms of the resulting separated analytes aredemonstrated in FIGS. 1A-M for cortisol, cortisone, corticosterone,11-deoxycortisol, testosterone, DHEA, deoxycorticosterone,androstenedione, 17-OH progesterone, progesterone, dihydrotestosterone,pregnenolone, and 17-OH pregnenolone, respectively. It should be notedthat in FIGS. 1H and 1I for androstenedione and 17-OH progesterone,respectively, an erroneous chromatographic peak was observed. Thesepeaks are labeled in the Figures with an X.

These separated samples were then subjected to MS/MS for quantitation ofselected CAH panel analytes.

Example 2: Quantitation of CAH Panel Analytes by MS/MS

MS (and MS/MS) was performed on separated samples generated above byfirst generating ions from the sample. These ions were passed to thefirst quadrupole (Q1), which selected ions with a desired parent mass tocharge ratio. Ions entering Quadrupole 2 (Q2) collided with argon gas togenerate ion fragments, which were passed to quadrupole 3 (Q3) forfurther selection. Simultaneously, the same process using isotopedilution mass spectrometry was carried out with selected isotope-labeledinternal standards. All of the selected masses for each CAH panelanalyte are listed in Table 1, above.

FIGS. 2-14 show mass spectra resulting from fragmentation of theprecursor ions indicated in Table 1.

As seen in FIG. 2, exemplary MRM transitions that may be monitored forthe quantitation of cortisol include fragmenting a precursor ion with am/z of about 363.1±0.5 to product ions with m/z of about 121.0±0.5 and90.9±0.5. A fragment was also observed at a mass to charge ratio ofabout 327±0.5 that was not believed to be suitable for quantitation ofcortisol. This fragment is indicated in FIG. 2 with an X.

As seen in FIG. 3, exemplary MRM transitions that may be monitored forthe quantitation of cortisone include fragmenting a precursor ion with am/z of about 361.1±0.5 to product ions with m/z of about 163.2±0.5 and105.1±0.5.

As seen in FIG. 4, exemplary MRM transitions that may be monitored forthe quantitation of corticosterone include fragmenting a precursor ionwith a m/z of about 347.2±0.5 to product ions with m/z of about121.1±0.5 and 91.1±0.5. Two fragments were also observed at mass tocharge ratios of about 329±0.5 and 293±0.5 that were not believed to besuitable for quantitation of corticosterone. These fragments areindicated in FIG. 4 with an X.

As seen in FIG. 5, exemplary MRM transitions that may be monitored forthe quantitation of 11-deoxycortisol include fragmenting a precursor ionwith a m/z of about 347.1±0.5 to product ions with m/z of about109.1±0.5 and 97.1±0.5.

As seen in FIG. 6, exemplary MRM transitions that may be monitored forthe quantitation of testosterone include fragmenting a precursor ionwith a m/z of about 289.1±0.5 to product ions with m/z of about109.0±0.5 and 97.0±0.5.

As seen in FIG. 7, exemplary MRM transitions that may be monitored forthe quantitation of DHEA include fragmenting a precursor ion with a m/zof about 253.1±0.5 to product ions with m/z of about 197.1±0.5 and157.1±0.5.

As seen in FIG. 8, exemplary MRM transitions that may be monitored forthe quantitation of deoxycorticosterone include fragmenting a precursorion with a m/z of about 331.2±0.5 to product ions with m/z of about109.5±0.5 and 97.1±0.5.

As seen in FIG. 9, exemplary MRM transitions that may be monitored forthe quantitation of androstenedione include fragmenting a precursor ionwith a m/z of about 287.1±0.5 to product ions with m/z of about109.1±0.5 and 91.1±0.5.

As seen in FIG. 10, exemplary MRM transitions that may be monitored forthe quantitation of 17-OH progesterone include fragmenting a precursorion with a m/z of about 331.0±0.5 to product ions with m/z of about109.0±0.5 and 96.9±0.5.

As seen in FIG. 11, exemplary MRM transitions that may be monitored forthe quantitation of progesterone include fragmenting a precursor ionwith a m/z of about 315.2±0.5 to product ions with m/z of about109.1±0.5 and 97.1±0.5.

As seen in FIG. 12, exemplary MRM transitions that may be monitored forthe quantitation of dihydrotestosterone include fragmenting a precursorion with a m/z of about 273.2±0.5 to product ions with m/z of about105.1±0.5 and 91.1±0.5.

As seen in FIG. 13, exemplary MRM transitions that may be monitored forthe quantitation of pregnenolone include fragmenting a precursor ionwith a m/z of about 299.2±0.5 to product ions with m/z of about105.6±0.5 and 91.1±0.5.

As seen in FIG. 14, exemplary MRM transitions that may be monitored forthe quantitation of 17-OH pregnenolone include fragmenting a precursorion with a m/z of about 297.2±0.5 to product ions with m/z of about105.6±0.5 and 91.1±0.5.

As can be seen in the product ion scans in FIGS. 2-14, several otherproduct ions are generated upon fragmentation of the indicated precursorions. Any of the additional product ions indicated in FIGS. 2-14 may beselected to replace or augment the exemplary fragment ions describedabove and in Table 1.

Linearity studies were conducted for detection of cortisol,11-deoxycortisol, androstenedione, deoxycorticosterone,17-OH-progesterone, 17-OH-pregnenolone, testosterone, progesterone,pregnenolone, and DHEA across a range of concentrations for each analyteof approximately 0 ng/dL to 1000 ng/dL. Results of these studies arepresented in FIGS. 3 A-J, respectively.

The limits of quantitation of the CAH panel analyte were determined (foreach individual analyte except deoxycorticosterone, and for each analyteas part of a 10 member panel). Results of these studies are presented inTable 3, below.

TABLE 3 Limits of Quantitation for CAH Panel Analytes, Individually andWithin a Panel Analyte Individual LOQ (ng/dL) Panel LOQ (ng/dL) cortisol100.0 50.0 11-deoxycortisol 20.0 20.0 testosterone 2.0 10.0 DHEA 10.015.0 deoxycorticosterone — 25.0 androstenedione 5.0 10.0 17-OHprogesterone 8.0 20.0 progesterone 10.0 10.0 pregnenolone 5.0 15.0 17-OHpregnenolone 6.0 15.0

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount of17-OH pregnenolone in a sample by mass spectrometry, the methodcomprising: (i) purifying the sample by liquid chromatography; (ii)ionizing the sample under conditions suitable to produce one or more17-OH pregnenolone ions detectable by mass spectrometry, wherein the oneor more 17-OH pregnenolone ions comprise one or more ions having a m/zof about 297.2±0.5, 105.6±0.5, and/or 91.1±0.5; (iii) determining theamount of the one or more 17-OH pregnenolone ions by mass spectrometry,wherein the method has a limit of quantitation of 6 ng/dL; and (iv)using the determined amount of the one or more ions to determine theamount of 17-OH pregnenolone in the sample.
 2. The method of claim 1,wherein said liquid chromatography is high performance liquidchromatography, ultra high performance liquid chromatography, orturbulent flow liquid chromatography.
 3. The method of claim 1, whereinthe sample has been purified by solid phase extraction prior toionization.
 4. The method of claim 1, further comprising adding internalstandard to the sample, wherein an internal standard is used fordetermination of the amount of 17-OH pregnenolone in the sample.
 5. Themethod of claim 4, wherein the internal standard comprises anisotopically labeled analog of 17-OH pregnenolone.
 6. The method ofclaim 1, wherein the sample comprises plasma or serum.
 7. The method ofclaim 1, wherein the ionizing comprises ionization by electrosprayionization (ESI).